Electronic imaging systems are known for enabling imaging of animals, for example mice. An exemplary electronic imaging system 10 is shown in FIGS. 1A, 1B, 1C, and 1D. An example of this system is the KODAK In-Vivo Imaging System FX Pro. System 10 includes a light source 12; a sample environment 14 which allows access to the object or objects being imaged; an optically transparent platen 16 disposed within sample environment 14; an epi-illumination delivery system comprised of fiber optics 18 which are coupled to light source 12 and direct conditioned light (of appropriate wavelength and divergence) toward platen 16 to provide bright-field or fluorescence imaging; an optical compartment 20 which includes a mirror 22 and a lens and camera system 24; a communication and computer control system 26 which can include a display device, for example, a computer monitor; a microfocus X-ray source 28; an optically transparent planar animal support member 30 on which objects may be immobilized and stabilized by gravity; and a high-resolution phosphor screen 32, adapted to transduce ionizing radiation to visible light by means of high-resolution phosphor sheet 34, which is proximate to animal support member 30 and removable along direction indicated by arrow 36, by conventional means such as a motor and lead screw arrangement, not illustrated. In the illustrated imaging system, lens and camera system 24 are located below support member 30; however, those skilled in the art understand that the system could be reconfigured to provide for imaging from above the support member or from any suitable angle.
Light source 12 can include an excitation filter selector for fluorescence excitation or bright-field color imaging. Sample environment 14 is preferably light-tight and fitted with light-locked gas ports for environmental control. Such environmental control might be desirable for controlled X-ray imaging or for life-support of particular biological specimens. Imaging system 10 can include an access means or member 38 to provide convenient, safe and light-tight access to sample environment 14. Access means are well known to those skilled in the art and can include a door, opening, labyrinth, and the like. Additionally, sample environment 14 is preferably adapted to provide atmospheric control for sample maintenance or soft X-ray transmission (e.g., temperature/humidity/alternative gases and the like). Camera and lens system 24 can include an emission filter wheel for fluorescent imaging. Examples of electronic imaging systems capable of multimodal imaging are described in the previously mentioned, copending, commonly assigned applications of Vizard et al, Feke and Feke et al.
In operation, the system is configured for a desired imaging mode chosen among the available modes including X-ray mode, radioactive isotope mode, and optical imaging modes such as bright-field mode, fluorescence mode, luminescence mode, and an image of an immobilized subject, such as a mouse 40 under anesthesia and recumbent upon optically transparent animal support member 30, is captured using lens and camera system 24. System 24 converts the light image into an electronic image, which can be digitized. The digitized image can be displayed on the display device, stored in memory, transmitted to a remote location, processed to enhance the image, and/or used to print a permanent copy of the image. The system may be successively configured for capture of multiple images, each image chosen among the available modes, whereby a synthesized image, such as a composite overlay, is generated by the combination of the multiple captured images.
Mouse 40 may successively undergo craniocaudal rotation and immobilization directly onto planar animal support member 30 in various recumbent body postures, such as prone, supine, laterally recumbent, and obliquely recumbent, whereby the mouse is stabilized by gravity for each body posture, to obtain multiple views, for example ventral and lateral views as described in “Picture Perfect: Imaging Gives Biomarkers New Look”, P. Mitchell, Pharma DD, Vol. 1, No. 3, pp. 1-5 (2006). As seen in FIG. 1D, a plurality of animals 40 may be imaged at the same time, only one being shown for ease of illustration. Animals may be rotated manually about their craniocaudal axes to provide different viewing angles, as indicated by arrow 42.
Direct immobilization of the animal in a recumbent posture onto an optically transparent planar support and imaging from below is advantageous for several reasons. First, the range of craniocaudal rotation angles, and hence the range of view angles, is unlimited and continuous. Second, the human experimenter has access to the animal. For example, where an experiment requires an intimate delivery, such as by injection, oral gavage, inhalation, transrectal delivery, transdermal delivery, or transmucosal delivery, of a substance, such as a drug, optical fluorescence imaging agent, X-ray contrast agent, or radionuclide imaging agent, to an animal, it is often desirable to capture images of the animal both prior to and after delivery of the substance without substantially physically disturbing the animal in the imaging system. This is especially desirable when studying the perfusion and clearance of imaging or contrast agents or therapeutic response of a drug over time. Third, the human experimenter has access to the immediate environment around the animal. For example, it is often desirable to clean the immediate environment of residue, including animal urine, feces, or surface debris that might otherwise cause imaging artifacts. Fourth, by virtue of stabilization by gravity, minimal manipulation and restraint of the animal is required to place it in the imaging system in a desired posture, hence facilitating an ergonomic protocol for the human experimenter. Fifth, recumbent postures are desired to minimize physiological stress on the animal. Sixth, the optically transparent support provides a physical surface to serve as a reference for a focal plane for the lens and camera system to facilitate the capture of sharp, well-resolved images. Seventh, the optically transparent support facilitates multimodality imaging in that a removable phosphor screen can be located proximally to the support surface, therefore providing a common focal plane for both the optical imaging modes (bright-field mode, fluorescence mode, and luminescence mode) and the imaging modes requiring a phosphor screen (X-ray mode and radioactive isotope mode); the common focal plane is necessary for precise co-registration of overlaid images from the optical imaging modes and the imaging modes requiring a phosphor screen. Eighth, the imaging light path is stationary and common for all view angles and all imaging modes, thereby providing for simple, inexpensive components to define the imaging light path.
On the other hand, direct immobilization of a recumbent animal onto a planar support is disadvantageous in that in known imaging systems a means is lacking to precisely control the craniocaudal rotation angle, and hence the view angle, of the animal, for example to ±5 degree precision. Improved control of the view angle would improve the experimenter's ability to quantify molecular signals. In the case of optical molecular signals obtained from fluorescence and luminescence modes, the signal is dependent upon the depth of tissue between the animal surface and the distributed fluorescent or luminescent content inside the animal through which the light must travel. That depth of tissue is dependent upon the animal's posture. In the case of radioactive isotope molecular signals, the signal is dependent upon the distance of the distributed radioactive isotope inside the animal from the phosphor screen. That distance is dependent upon the animal's posture.
Improved control of the view angle also would improve the experimenter's ability to reproduce the spatial orientation of an animal. For example, an animal used in a longitudinal imaging study is loaded into an imaging system and immobilized for a first time, imaged for the first time, unloaded from the imaging system, loaded into the imaging system and immobilized for an at least second time, and imaged for the at least second time, thereby producing a first-time set of images and an at least second-time set of images. If the spatial orientation of the animal, for example the craniocaudal rotation angle of the animal, with respect to the imaging system is different between the first time and the at least second time, then the at least second-time set of images may be affected by the difference in the spatial orientation compared to the first-time set of images. This difference may result in artifacts, such as relative attenuation or enhancement of a molecular signal, upon comparison to the first-time set of multimodal molecular images. Similarly, this difference may lead to a result that the first-time set of images and the at least second-time set of images will not be co-registered, thereby degrading the quantitation provided by a simple regions-of-interest analysis wherein a single regions-of-interest template is applied to both the first-time set of images and the at least second-time set of images.
For example, when a plurality of animals are used in an imaging study, and are loaded into an imaging system and immobilized, whereby the loading and immobilization may be performed serially at a given spatial location within the field of view of the imaging system, or may be performed in parallel across a plurality of spatial locations in the field of view of the imaging system, the spatial orientations of the animals, for example the craniocaudal rotation angles, may differ among the plurality of animals, so that each set of images for each animal may be affected by the difference in the spatial orientation, thereby resulting in artifacts, such as relative attenuation or enhancement of a molecular signal, in one set of images compared to another set of images.
If animals are loaded serially at a given spatial location within the field of view, then the sets of images may not be co-registered among the plurality of animals due to differences in the spatial orientations of the animals, for example the craniocaudal rotation angles, thereby degrading quantitation provided by a simple regions-of-interest analysis wherein a single regions-of-interest template is applied to the sets of images corresponding to the plurality of animals.
If animals are loaded in parallel across a plurality of spatial locations in the field of view of the imaging system, then regions of interest defined for one animal may not be spatially translatable to the other animals by the simple difference between the spatial locations of the animals due to differences in the spatial orientations, for example the craniocaudal rotation angles, of the animals at their locations, thereby degrading quantitation provided by a simple regions-of-interest analysis wherein an array-like regions-of-interest template (i.e., multiple copies of a set of regions of interest across the field of view) is applied to the set of images.
Although imaging systems are known that can obtain multiple views of an animal with a stationary camera system wherein the view angle is more precisely controlled, none of these provides all of the advantages of direct immobilization of a recumbent animal onto a support. Strapping the subject down onto an angularly adjustable goniometer stage and imaging from above is described in “Factors Influencing Quantification of In Vivo Bioluminescence Imaging: Application to Assessment of Pancreatic Islet Transplants”, J. Virostko et al., Molecular Imaging, Vol. 3, No. 4, pp. 333-342 (2004). Suspending the animal in a holder which is clamped onto a rotation stage is described in “Systems and Methods for Bioluminescent Computed Tomographic Reconstruction”, Wang et al., U.S. patent application Ser. No. 10/791,140, Publication US2004/0249260. Placing the subject into a rotatable tube is described in “System and Method for Visualizing Three-dimensional Tumor Locations and Shape from Two-dimensional Bioluminescence Images”, D. Metaxas et al., U.S. Provisional Patent Application Ser. No. 60/715,610, Publication WO/2007/032940; and in “Design of a Small Animal Multimodality Tomographer for X-Ray and Optical Coupling: Theory and experiments”, da Silva et al., Nuclear Instruments and Methods in Physics Research A, Vol. 571, Nos. 1-2, pp. 118-121, (2007). Employing a rotating mirror and a translating subject stage is described in “Multi-view imaging apparatus”, D. Nilson et al., U.S. Pat. No. 7,113,217. Employing a multiple mirror assembly that surrounds a portion of the subject to direct additional views to the camera system is described in “Systems and methods for in-vivo optical imaging and measurement”, R. Levenson and C. Hoyt, U.S. patent application Ser. No. 11/295,701, Publication US2006/0118742.
While systems of the types described in the just-mentioned publications and patents may have achieved certain advantages, each of them also exhibits one or more of the following disadvantages. The adjustable stage has a limited angular range thereby limiting the range of view angles. The animal holder obstructs the view for certain view angles. The human experimenter has limited access to the immediate environment around the animal. Significant manipulation of the animal is required to strap it down to prevent sliding on the adjustable stage, to suspend it in the holder or to load it into a tube in a desired posture. A physical surface to serve as a reference for a focal plane is not provided. A surface for the proximal location of a phosphor screen is not provided. The animal is not recumbent, hence it is subject to significant physiological stress. The imaging light path is different for every view angle, thereby requiring complex, expensive components to define the imaging light path. A multiple mirror assembly has a limited, discontinuous angular range, thereby limiting the range of view angles.
Imaging systems also are known that can obtain multiple views of an animal, comprised of one or more lens and camera systems mounted on a gantry that may be rotatable about the animal. For example, see “Method and system for free space optical tomography of diffuse media”, V. Ntziachristos and J. Ripoll, U.S. patent application Ser. No. 10/543,728 and U.S. Patent Application Publication 2006/0173354; and “Combined X-ray and Optical Tomographic Imaging System”, W. Yared, U.S. patent application Ser. No. 11/643,758 and U.S. Patent Application Publication 2007/0238957. However, such systems are significantly more complex and expensive compared to a single, stationary imaging light path common for all view angles and all imaging modes.
In addition, photoacoustic tomography systems are known for enabling photoacoustic tomography of animals, for example mice. An exemplary photoacoustic tomography system is described in “HYPR-spectral photoacoustic CT for preclinical imaging”, Kruger et al., Proc. SPIE, Vol. 7177, 71770F (2009). The system is comprised of an array of ultrasound sensors disposed in a bowl. The bowl has an optically transparent window in the bottom. In operation, the bowl is filled with an optically transparent acoustic coupling medium, such as a liquid or gel, an optically transparent membrane is placed at the top surface of the acoustic coupling medium, the animal is immobilized and placed on the membrane in a recumbent posture, pulsed light is provided through the window to the animal, the light is absorbed by endogenous and/or exogenous material in the animal, the material releases energy as ultrasound, the ultrasound is detected by the array of sensors, and a electronic system performs a tomographic reconstruction based on the detected ultrasound. Because the penetration depth of the light is limited by absorption and scatter in the animal, it is desirable to be able to position the animal in a variety of postures to access the various anatomical features of the animal. In known photoacoustic tomography systems, animals may be rotated manually about their craniocaudal axes to provide different viewing angles. However, direct immobilization of a recumbent animal onto a membrane is disadvantageous in that in known photoacoustic tomography systems a means is lacking to precisely control the craniocaudal rotation angle, and hence the view angle, of the animal, for example to ±5 degree precision. Improved control of the view angle would improve the experimenter's ability to quantify photoacoustic data. For example, photoacoustic calibrations that depend on the tissue depth, and hence the animal posture, could be more precisely determined. Also, different photoacoustic tomographic sections from a series of different view angles could be precisely stitched together given precise knowledge of the view angle.